One of the goals of the Bahia field season was to look a little deeper at the interaction between kleptoplasty and chemical defense.  The general idea went something like this:

1. Kleptoplasts continue to photosynthesize after they take up residence in the slugs’ tissues.
2. Slugs produce defensive chemicals that make them taste bad.
3. Kleptoplasts could be the source of the defensive chemicals: is photosynthesis required to make them?

Based on this line of thinking, we hypothesized that slugs deprived of light should be less distasteful than those kept in bright light.

To put the hypothesis to the test, we set up two tanks that were nearly identical except for lighting.  They were plumbed into the same sump and chiller, so their temperatures and chemistry were essentially identical, but one was illuminated by a high-intensity LED fixture to support photosynthesis (PAR ≥100 mol m⁻²s⁻¹), while the other was shaded to reduce the light by 100-fold (PAR < 1 mol m⁻²).

Tanks ready for slugs. Each contains a nearly identical collection of rocks and algae. The tank on the right is illuminated by a high-output LED fixture, while the one on the left is shaded by black felt, resulting in a 100-fold difference in intensity. 6/30/18.

Once we obtained the permit and were allowed to collect, we randomly separated slugs into two tanks that contained roughly equal amounts of Codium on which they could feed.  They were allowed to feed at will, because the goal was to test the role of photosynthesis in generating defensive chemicals, not the effect of starvation.

The original plan was to use mucus from experimental (unlit) and control (lit) slugs to make food cubes, then test which ones were eaten by fishes in the bay.  However, based on experiments at USG, and the fact that we would not be able to extract enough mucus from our few dozen slugs, we decided to test the effects of tissue extracts from whole slugs.  Surprisingly, the students were not as sad as we might have expected that they had to purée their pets.

For the description below, whenever I write “we,” I really mean Ric and the students, because he was very much in charge of this project.  Most of the below photos are his.

Zaira weighing carrageenan for a batch of food cubes. 7/3/18.

The process was as quirky as any of the other experiments we have done in Bahia.  To make the gel base of the food cubes, we needed to dissolve carrageenan in water, which required heating the water to boiling.  The easiest method is to microwave the water, but the only available microwave oven had a single working button (“popcorn”), which turned the microwave on (after a disconcerting delay) for 3.5 minutes.  It took some practice and finesse to turn the machine off at the right time, but they managed.

Zaira stirring food and carrageenan mix. Dodgy microwave oven is at right, and some of the molecular crew (Lili, Dave, Elizabeth) are in the background. 7/3/18.

Fish food pellets were ground and added to the carrageenan solution to make the base food cubes.  Mucus or tissue were then added to a portion of the food, depending on the experiment.

Food mixed and ready to pour. 7/3/18.

Once heated and thoroughly mixed, food was poured into silicone ice cube trays to make 1 cm cubes.  Silicone O-rings, used to secure the food cubes to clips during experiments, were placed into the molds and held steady with acrylic rods.

Bennie setting up rings in food forms, getting ready to pour how food mix. 7/3/18.

Once the O-rings were in place, food was poured into the molds and evened out using a knife.  After cooling for a bit, the whole assembly was stuffed into a ziploc bag and refrigerated until needed.

Food cubes poured and ready for use. Note acrylic rods to keep rings in place. 7/3/18.

At experiment time, food cubes were attached to monofilament lines to be anchored in the bay.  The bottom end of the line was tied to small lead weights, while the top was held afloat by a 16-20 oz soda bottle.  Food cubes were secured by O-rings to plastic safety pins tied to the line at regular intervals.  Several bugs needed to be worked out.  For example, we started with lightweight fishing line, but it had a tendency to break in the surge of the intertidal zone, causing loss of the whole bait line.  We shifted to a much heavier gauge, which apparently scared the fish away, because all food cubes were present after 24 hours.  Ultimately, we settled on something in between, and could start gathering real data.

Keyla, demonstrating system for placing food cubes in the bay.  Plastic safety pins are tied to monofilament fishing line, which is suspended between the float in her right hand and the weights by her left foot.  Food cubes are then clipped to safety pins via embedded O-rings.  7/13/18.

Once food cubes were secure, the lines were placed deep enough to keep them afloat at low tide.

Food cubes suspended in water column in bay.  7/13/18.

Lines were collected after a predetermined time (see below).

Collecting line of now-empty clips.  7/13/18.

For the first round of experiments, cubes were left in the bay for 24 hours.  During this time, essentially all of the control cubes (i.e., without tissue or mucus) were eaten, as were most of the experimental cubes.  Nonetheless, fish appeared to eat fewer cubes containing tissue from slugs kept in the light, compared to those kept in the dark.

Effect of food contents on consumption by fish in the bay. When collected, each line was given a score, with 1 = all cubes intact, and 3 = all cubes gone. In this experiment, all control cubes were missing (and presumed consumed) in 24 hours, as were cubes made from tissue prepared from slugs kept in the dark for one week (right most bar). Food made from slugs kept in light (second from right) had a lower score than the other groups, but the difference was not significant.  Sample sizes: 18 control, 12 mucus, 12 lit, 12 dark.

Because there was concern that missing cubes may have fallen off rather than being eaten, they also tried leaving the cubes in the bay for only one hour.  There was a slight decrease in consumption, but the overall result was the same: tissue from dimly-lit slugs was eaten more often than that from well-lit slugs.  The students were running out of time for these experiments, so the results are preliminary and nothing is statistically significant.

Results of leaving the food lines in the bay for an hour. Food made from slugs kept in the light (middle bar) had a lower consumption score than those without slug tissue (left bar) or made from slugs kept in dim light (right bar). Sample sizes: 18 control, 24 lit, 24 dark.

To summarize several weeks of hard work, it looks like there is something to pursue.  The sample sizes are small, but there is a consistent effect of slug tissue in reducing consumption by fishes, and this effect is reduced or eliminated by keeping slugs in the dark.  Now that the bugs have been worked out, it will certainly be worth trying the experiment again if there is an opportunity to spend a few weeks making food and setting lines.

Thanks again to the USG Slug Club, who pioneered the foodmaking methods, and took care of the slug system while I was away.